Project Summary Enveloped viruses enter cells using fusion proteins to merge the virus envelope and the cell membrane. Determining the atomic-level structures and structural changes of viral fusion proteins along the fusion pathway is important both for fundamental understanding of how proteins mediate membrane-curvature generation and for potentially developing antiviral drugs to inhibit virus entry. High-resolution structural information of the membrane-bound fusion peptide (FP) and transmembrane (TM) domains in viral fusion proteins is so far limited. We propose to investigate the FP and TM domains of the HIV and parainfluenza virus 5 (PIV5) fusion proteins, using high-resolution solid-state NMR spectroscopy. Aim 1 will examine the oligomeric structure of the PIV5 FP and TM peptides in lipid membranes of different compositions to understand early-fusion structures. In Aim 2, we will investigate the structure of a fusion protein chimera that links the FP and TM segments, in order to understand late-fusion structures. By measuring inter-domain FP-TM contacts and homo-oligomeric association, we will determine whether the FP and TM form a six-helix bundle in the lipid membrane, similar to the ectodomain six-helix bundle outside the membrane. In Aim 3, we will investigate the MPER- TM structure of the HIV fusion protein, gp41. We will conduct 19F-19F and 13C-19F distance experiments to measure the three-dimensional fold and oligomeric structure of this antibody- targeted region of the protein. These experiments should provide fundamental insights into the protein structural transitions that underlie virus-cell fusion.